Application of corn Zm00001d016228 gene or encoded protein thereof in regulation and control of grain traits

Abstract

The invention discloses an application of a corn Zm00001d016228 gene or an encoded protein thereof in regulation and control of grain traits, the hundred-grain weight of corn is an important factor influencing the yield and the seed yield of corn, and along with the increase of the hundred-grain weight of corn, the higher the seed yield is, the higher the yield is. According to experiments, the Zm00001d016228 gene is over-expressed in corn, the hundred-grain weight is remarkably increased, a good theoretical basis and a good theoretical direction are provided for genetic improvement of the hundred-grain weight and cultivation of high-seed-yield characters and high yield, and the Zm00001d016228 gene has reliable prospects when applied to the biological breeding process of corn kernel character improvement.

Description

Technical Field

[0001] The invention relates to the technical field of corn molecular breeding, and in particular to the application of corn Zm00001d016228 gene or its encoded protein in regulating grain traits. Background Art

[0002] Corn is an important food, feed and energy crop. Achieving large-scale yield increase is of great significance to ensuring national food security. Grain traits, including grain length, grain width, and 100-grain weight, are important yield components and play an important role in yield trait improvement. Corn grain traits are typical quantitative traits and are easily affected by environmental conditions. The selection of germplasm resources through traditional breeding methods has problems such as long cycle and low efficiency. Therefore, discovering new genes that control corn grains, deeply understanding the molecular mechanism of corn grain development, and analyzing the regulatory mechanism of corn grain development from a genetic perspective can provide more and better breeding schemes for corn breeding, shorten the breeding cycle, and improve breeding efficiency. Therefore, discovering key genes that control corn grain development is not only of theoretical significance, but also has broad application prospects. The corn Zm00001d016228 gene encodes a protein containing an NHL domain, which can be obtained through QTL positioning. At present, the role of the Zm00001d016228 gene in regulating 100-grain weight has not been reported. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides the application of the corn Zm00001d016228 gene or its encoded protein in regulating grain traits. This gene has important theoretical significance and practical application value in improving corn grains and cultivating new high-yield corn varieties, and provides a new direction for the subsequent cultivation of new high-yield corn lines and the acquisition of germplasm resources.

[0004] The first technical solution of the present invention provides: Zm00001d016228 gene related to corn grain traits, the nucleotide sequence of the coding region of the Zm00001d016228 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Zm00001d016228 gene is shown in SEQ ID NO.2.

[0005] Furthermore, the corn kernel traits include corn 100-kernel weight.

[0006] The second technical solution of the present invention provides the use of the Zm00001d016228 gene related to corn kernel traits, or a product overexpressing the Zm00001d016228 gene in regulating corn kernel traits.

[0007] Furthermore, the regulation refers to overexpressing the Zm00001d016228 gene in corn materials to improve corn grain traits.

[0008] Furthermore, the product overexpressing the Zm00001d016228 gene is one of the following:

[0009] ① an expression cassette containing the Zm00001d016228 gene;

[0010] ② A recombinant expression vector containing the Zm00001d016228 gene;

[0011] ③ A genetically engineered bacterium containing the Zm00001d016228 gene.

[0012] The third technical solution of the present invention provides the application of the Zm00001d016228 gene and the protein encoded by it, or the product overexpressing the Zm00001d016228 gene, and the application includes at least one of the following:

[0013] 1) Application in improving corn kernel traits;

[0014] 2) Application in corn breeding or assisted breeding.

[0015] The fourth technical solution of the present invention provides the use of the Zm00001d016228 gene or its encoded protein in identifying or breeding or assisting in breeding corn plants, lines or varieties with excellent grain traits.

[0016] The fifth technical scheme of the present invention provides a method for cultivating high 100-grain weight corn, wherein the corn gene Zm00001d016228 is transformed into corn immature embryos, and then the corn transgenic strain overexpressing the Zm00001d016228 gene is obtained after cultivation.

[0017] Furthermore, the obtained transgenic strains overexpressing the maize gene Zm00001d016228 are subjected to continuous self-pollination or hybridization to obtain homozygous offspring that stably overexpress the Zm00001d016228 gene.

[0018] The sixth technical solution of the present invention provides a breeding method for high 100-grain weight corn, in which at least one parent overexpresses the Zm00001d016228 gene for hybrid breeding.

[0019] The beneficial effects achieved by the present invention are as follows:

[0020] Corn 100-grain weight is an important factor affecting corn yield and seed rate. As corn 100-grain weight increases, the seed rate increases and the yield increases. The present invention has been experimentally verified to significantly increase 100-grain weight by overexpressing the Zm00001d016228 gene in corn, providing a good theoretical basis and direction for genetic improvement of 100-grain weight and cultivating high seed rate traits and high yields, and applying it to the biological breeding process of corn seed trait improvement has a reliable prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 This is the positive identification of the gene overexpression plants in the examples of the present invention.

[0022] Attached Figure 2 This is a grain comparison diagram of the overexpression mutant of the present invention and the B73 inbred line in the examples of the present invention.

[0023] Attached Figure 3 Statistical diagram of grain phenotype of overexpression mutant lines. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following implementations, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0026] Example 1: Construction and identification of Zm00001d016228 gene overexpression strain

[0027] 1. Construction of Zm00001d016228 gene overexpression vector

[0028] The CDS sequence of Zm00001d016228 gene (SEQ ID No. 1, 1548 bp) was cloned from B73 leaves, and it was found that the gene encodes a protein consisting of 516 amino acids (SEQ ID No. 2).

[0029] The CDS sequence of the gene was transferred into the plant overexpression vector 35s-pMDC83-GFP-Gate using the LR reaction of the Gateway system. The recombinant vector pMDC83-Zm00001d016228 containing the 13S strong promoter was transformed into Agrobacterium AGL1. In cooperation with Weimi Company, B73 transgenic materials were created, and T0 generation overexpression transgenic seedlings were obtained. The DNA of the T0 generation transformed plants was extracted and the genotype was identified.

[0030] The CDS sequence of Zm00001d016228 obtained by cloning (SEQ ID No. 1)

[0031]

[0032] Amino acid sequence of the protein encoded by Zm00001d016228 (SEQ ID No. 2)

[0033] MGLRSPLVLLLLLLLFLAATFRPSAAALAPPTGSIVKQLSSVVRWPRAAPSTHGPKQPGHPQY

[0034] ADGHVGVALQFESGYFVETLVEGDKLGVTPHTVRVSPVEGGELLAVDSAHSNIVRITPPLSE

[0035] YSRGRLVAGSFQGHSGHIDGKPSDARFKRPTGVAVDDMGNVYVADTANLAIRKIGESGVTTI

[0036] AGGKSNIPGYRDGPSEDAKFSTDFDVVYVKKMCSLLVIDRGNAALRKISLPQEDCTYQDSA

[0037] LLSSVPYFIINFVLFIYIDLILVIGAVVAGYIFSGFQHGFGFSGSEKVEAPENEQHESSTIGKPPL

[0038] VVESLKEEPGAGWPSLGTLIADLLKLAIEGVGKLLLSVVPQRMQHGKRKTDLTPLRDRLVM

[0039] PEDREETAAAAQKLSSTPMRPETAHAPNPVTETAAPKAPKSTKPSKLRDSSTLSSKHRSSKR

[0040] QEYADFYGTSEPAPVGAAAKVPKDRLRHRHHHREKSGEVAYGAAHHDLKPAEAKPADYSDPSYDPYVRSKYAAESGYRY.

[0041] 2. Identification of overexpression lines of Zm00001d016228 gene

[0042] The DNA of Zm00001d016228 transgenic maize plants was extracted using the CTAB method. The specific steps are as follows:

[0043] (1) Take about 200 mg of fresh leaves and put them into a 2 ml centrifuge tube. Grind them into powder using liquid nitrogen cryo-grinding or a grinder.

[0044] (2) Add 600 μl of 2× CTAB preheated at 65°C, mix well, and keep in a 65°C water bath for 20 min. During this time, shake up and down 2-3 times to ensure complete cell lysis.

[0045] (3) Add 600 μl of chloroform / isoamyl alcohol (24:1), mix by inverting for 1 min, and then let stand for 3-5 min to allow the layers to separate.

[0046] (4) Centrifuge at 10,000 rpm for 10 min and carefully transfer the supernatant to a new 1.5 ml centrifuge tube.

[0047] (5) Add an equal volume of -20°C precooled isopropanol, mix by inverting, and place at -20°C for 30 min to precipitate the DNA.

[0048] (6) Centrifuge at 12,000 rpm for 10 min, discard the supernatant, add 0.5 ml of 70% ethanol solution, and wash the DNA precipitate by inverting it (if the DNA precipitate slides or floats, centrifuge at 12,000 rpm for 5 min after washing to allow the DNA to adhere to the bottom of the tube). Discard the ethanol solution (be careful not to discard the white DNA precipitate at the bottom of the centrifuge tube).

[0049] (7) After pouring off the ethanol, dry the DNA precipitate at room temperature.

[0050] (8) Add 200 μl of pure water to dissolve the DNA and store at 4°C until use.

[0051] The 2×CTAB solution was prepared as follows:

[0052] CTAB powder: 4 g, NaCl: 16.364 g, 1M Tris-HCl: 20 ml (PH = 8.0), 0.5 M

[0053] EDTA: 8 ml, dissolve in ultrapure water, make up to 200 ml, sterilize under high pressure and add mercaptoethanol.

[0054] The obtained genomic DNA was amplified by PCR using detection primers. 2 μl of transgenic corn DNA was used as a template to detect the primers:

[0055] F: 5-CCATCGTCAACCACTACATCGAGACA-3' (SEQ ID NO: 4)

[0056] R: 5'-CTTCAGCAGGTGGGTGTAGAGCGT-3' (SEQ ID NO: 5)

[0057] PCR amplification was performed under the following conditions: preheating at 95°C for 5 min; 25-30 amplification cycles (denaturation at 98°C for 10 sec, annealing at 65°C for 30 sec, and extension at 72°C for 30 sec / kb), and extension at 72°C for 2 min.

[0058] The amplification system is: 5 μL 5×Q5 Reaction Buffer, 0.5 μL 10 mM dNTPs, 1.25 μL 10 μM primer, 0.25 μL Q5 High-Fidelity DNA Polymerase and template (1 ng~1 μg), and add ddH2O to make up to 25 μL.

[0059] The target fragment with a length of 1548 bp was amplified (see Figure 1 ), sequencing proved that the fragment was the recombinant vector NHL-Zm00001d016228 fragment, proving that the recombinant vector had been transferred into corn. According to the pure line calculation method, more than 10 plants were positive, indicating that the line was pure, so they were named NHL-OE1, NHL-OE2, and NHL-OE3.

[0060] Example 2: Verification of the effect of Zm00001d016228 gene overexpression on regulating 100-grain weight of corn

[0061] The three positive overexpression strains of the identified Zm00001d016228 gene, OE1, OE2, OE3, and the wild strain were planted in the experimental field of the Guizhou Dry Grain Research Institute, and harvested after maturity and the grain phenotypes were counted. The statistical data were analyzed using SPSS software, and the analysis method was one-way ANOVA.

[0062] The results are shown in Table 1.

[0063] Table 1 Statistics of 100-grain weight of different corn strains

[0064] strain 100-grain weight (g) B73 26.9 OE1 <![CDATA[35.5 * ]]> OE2 <![CDATA[31.5 * ]]> OE3 <![CDATA[29.5 * ]]>

[0065] Notes in the table: * indicates significant difference compared with wild-type B73.

[0066] As shown in Table 1, compared with the wild-type B73, the 100-grain weight of the Zm00001d016228 overexpression strain was significantly increased, ranging from 9.67 to 31.97%, indicating that Zm00001d016228 plays an important role in regulating the 100-grain weight of corn.

[0067] In summary, the combination of positive identification results and field phenotypic identification results showed that Zm00001d016228 could significantly increase the 100-grain weight of corn, and could be used for genetic improvement of 100-grain weight of corn and to increase seed yield and yield.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Zm00001d016228 gene associated with corn kernel traits, characterized in that: The nucleotide sequence of the coding region of the Zm00001d016228 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Zm00001d016228 gene is shown in SEQ ID NO.

2.

2. The Zm00001d016228 gene related to corn kernel traits according to claim 1, characterized in that: The corn kernel traits include corn 100-kernel weight.

3. Use of the Zm00001d016228 gene related to corn kernel traits as claimed in claim 1, or a product overexpressing the Zm00001d016228 gene, in regulating corn kernel traits.

4. The use according to claim 3, characterized in that: The regulation refers to overexpressing the Zm00001d016228 gene in corn materials to improve corn grain traits.

5. The use according to claim 3, characterized in that: The product overexpressing the Zm00001d016228 gene is one of the following: ① An expression cassette containing the Zm00001d016228 gene according to claim 1; ② A recombinant expression vector containing the Zm00001d016228 gene according to claim 1; ③ A genetically engineered bacterium containing the Zm00001d016228 gene described in claim 1.

6. The use of the Zm00001d016228 gene and the protein encoded by it according to claim 1, or the product overexpressing the Zm00001d016228 gene according to claim 5, characterized in that: The application includes at least one of the following: 1) Application in improving corn kernel traits; 2) Application in corn breeding or assisted breeding.

7. Use of the Zm00001d016228 gene or its encoded protein according to claim 1 in identifying or breeding or assisting in breeding of corn plants, lines or varieties with excellent grain traits.

8. A method for cultivating high 100-grain weight corn, characterized in that: After the maize gene Zm00001d016228 was transformed into maize immature embryos, a maize transgenic strain overexpressing the Zm00001d016228 gene was obtained after cultivation.

9. The method for cultivating high 100-grain weight corn according to claim 8, characterized in that: The obtained transgenic strains overexpressing the maize gene Zm00001d016228 are subjected to continuous self-pollination or hybridization to obtain homozygous offspring stably overexpressing the Zm00001d016228 gene.

10. A method for breeding high 100-kernel weight corn, characterized in that: Cross breeding was performed with at least one parent overexpressing the Zm00001d016228 gene.

Images